The intestinal immune system is charged with the difficult task of protecting a large environmentally exposed surface from potential pathogens, while simultaneously preventing inflammatory responses to innocuous foreign antigen from food and commensal microbiota. Failure to appropriately protect the mucosa can result in life-threatening enteric infection, and failure to control intestinal immune responses results in chronic debilitating disorders such as inflammatory bowel disease. Recent studies determined that the lamina propria (LP) DC population is primarily comprised of dichotomous (CD103+ tolerogenic or CX3CR1 + inflammatory) DCs. This discovery suggests that the balance between tolerance and immunity rests upon which LP DC subtype participates in the immune responses. However a key and missing component is in vivo knowledge of which LP DC subtypes acquire antigen, the anatomical and cellular context of the each LP DC subtypes'interactions with T cells and microbiota, and the outcomes of these interactions on the character of the cellular immune response. The studies outlined in this proposal will harness the complementary expertise of two laboratories to examine the pathways guiding the delivery of pathogenic and non-pathogenic antigens to LP DCs and the outcomes associated with antigen acquisition by LP DC subtypes. The proposed studies make extensive use of cutting-edge two-photon imaging technology to analyze the trafficking and function of DCs and T cells in the intestine of living mice. The overarching hypothesis of this proposal is that antigen acquisition pathways guide immune responses by delivering antigen to specific LP DC subtypes. The studies in Aim 1 will use complimentary in vivo and in vitro approaches to examine the pathways delivering antigen to LP DC subtypes in the uninfected and infected state. Aim 2 will evaluate LP DC subtype specific responses in the presence and absence of infection. Aim 3 will evaluate the capacity of the LP DC subtypes to shape pre- existing T cell mediated immune responses locally within the intestinal lamina propria. Completion of these studies will put forth a new paradigm demonstrating that antigen acquisition pathways are a controlled proximal mechanism guiding immune response toward tolerance or immunity. RELEVANCE: The intestinal immune system must protect us from a wide array of potential pathogens and simultaneously avoid over-exuberant responses resulting in chronic intestinal inflammation. This study will investigate a novel mechanism for maintaining the balance between immunity and tolerance and will offer new avenues to pursue for improved mucosal vaccine therapy and chronic intestinal inflammation.